基于CRISPR/Cas9技术验证人T淋巴细胞中单等位突变型UNC13D基因脱颗粒功能  

The degranulation function of monoallelic mutant UNC13D gene in human T lymphocytes by CRISPR/Cas9

作　　者：蔡丽莎 邢源 黄佩 陈艳 徐晓军[2] Cai Lisha;Xing Yuan;Huang Pei;Chen Yan;Xu Xiaojun(Department of Pediatrics,the Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China;Department of Hematology-Oncology,Children’s Hospital of Zhejiang University School of Medicine,Hangzhou Zhejiang 310003,China;Department of Pediatrics,Guizhou Children’s Hospital,Zunyi Guizhou 563099,China;Collaborative Center for Tissue Injure Repair and Regenerative,Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]遵义医科大学附属医院小儿内科,贵州遵义563099 [2]浙江大学医学院附属儿童医院血液肿瘤内科,浙江杭州310003 [3]贵州省儿童医院小儿内科,贵州遵义563099 [4]遵义医科大学组织损伤修复与再生医学省部共建协同创新中心,贵州遵义563099

出　　处：《遵义医科大学学报》2023年第11期1058-1066,共9页Journal of Zunyi Medical University

基　　金：浙江省自然科学基金资助项目(NO:LY19H080006)。

摘　　要：目的利用慢病毒介导的CRISPR/Cas9基因编辑技术,敲除人T淋巴细胞中编码Munc13-4蛋白的UNC13D基因;并探讨突变型UNC13D基因(c.1232 G>A,p.R411Q)对人T淋巴细胞Munc13-4蛋白和细胞毒性T淋巴细胞(CTL)脱颗粒功能的影响。方法设计靶向敲除UNC13D基因的sgRNA序列,构建CRISPR/Cas9 UNC13D-sgRNA共表达质粒,筛选出切割效率较高的sgRNA通过第二代慢病毒包装系统包装慢病毒并感染人T淋巴细胞,再将已包装好的野生型以及突变型UNC13D慢病毒感染knockout-UNC13D基因的人T淋巴细胞,以此分为野生型与突变型两组,应用流式细胞术检测两组间的Munc13-4蛋白表达、CTL的脱颗粒功能。结果成功构建出CRISPR/Cas9 UNC13D-sgRNA共表达质粒,CRISPR/Cas9系统成功下调人T淋巴细胞Munc13-4蛋白表达水平;突变型UNC13D基因(c.1232 G>A,p.R411Q)编码的蛋白能够使人CTL脱颗粒功能下降。结论CRISPR/Cas9基因编辑系统成功敲除人T淋巴细胞UNC13D基因,突变型UNC13D基因(c.1232 G>A,p.R411Q)可能为家族性噬血细胞综合征(FHL)的致病性突变位点。Objective To knock out UNC13D gene encoding Munc13-4 protein in human T lymphocytes by lentivirus-mediated CRISPR/Cas9 gene editing.To investigate the mutant UNC13D gene(c.1232 G>A,p.R411Q)effect on Munc13-4 protein of human T lymphocytes and degranulation function of cytotoxic T lymphocytes(CTL).Methods The sgRNA sequence targeting UNC13D gene knockout was designed to construct the CRISPR/Cas9 UNC13D-sgRNA co-expression plasmids.The sgRNA with higher cutting efficiency was screened out and packaged by the second-generation lentivirus packaging system and then infected human T lymphocytes.T lymphocytes with UNC13D knockout were infected with the packaged wild type and mutant UNC13D lentivirus.The expression of Munc13-4 protein and the degranulation function of CTL between the two groups were detected by Flow cytometry.Results Successfully constructed the CRISPR/Cas9 UNC13D-sgRNA co-expression plasmids and the CRISPR/Cas9 system successfully down-regulated Munc13-4 protein expression in human T lymphocytes.Protein that encoded by mutant UNC13D gene(c.1232 G>A,p.R411Q)can reduce the degranulation function of human CTL.Conclusion CRISPR/Cas9 system can successfully ablate UNC13D gene in human T lymphocytes.While the mutant UNC13D gene(c.1232 G>A,p.R411Q)may be a pathogenic mutation site of familial hemophagocytic lymphohistiocytosis(FHL).

关 键 词：CRISPR/Cas9系统 家族性噬血细胞综合征 基因编辑 UNC13D基因 T淋巴细胞 

分 类 号：R392.7[医药卫生—免疫学]